Combination chemotherapy

ABSTRACT

Acetyldinaline in combination with gemcitabine, a pharmaceutically acceptable salt thereof, capecitabine, or cisplatin is synergistic for treating cancer.

FIELD OF THE INVENTION

[0001] The invention concerns a method for treating tumors utilizing acombination of known oncolytic agents. The use of the agents togetherprovides unexpectedly greater efficacy than employing the single agentsalone.

BACKGROUND OF THE INVENTION

[0002] Cancer chemotherapy has advanced dramatically in recent years.Many tumors can be effectively treated utilizing compounds which areeither naturally occurring products or synthetic agents. Cancerchemotherapy often entails use of a combination of agents, generally asa means of providing greater therapeutic effects and reducing the toxiceffects that are often encountered with the individual agents when usedalone.

[0003] We have now discovered a unique combination of known oncolyticagents which exhibits a dramatic synergistic effect. The combinationutilizes the agent acetyldinaline, together with either gemcitabine,capecitabine, or cisplatin. The combination is especially effective intreating patients with solid tumors, especially nonsmall cell lungcancer and other advanced solid tumors.

[0004] Acetyldinaline is 4-acetylamino-N-(2′-aminophenyl)-benzamide. Itis also known as CI-994. It is described in U.S. Pat. No. 5,137,918,which is incorporated herein by reference for its teaching of how tomake acetyldinaline, how to formulate it into dosage forms, and how touse it for treating cancers such as colon cancer and adenocarcinomas. Itis also described in U.S. Pat. No. 5,795,909 as a possible conjugate forcancer treatment.

[0005] Gemcitabine is the generic name assigned to2′-deoxy-2′,2′-difluoro-cytidine. It is commercially available as themonohydrochloride salt, and as the β-isomer. It is also known chemicallyas 1-(4-amino-2-oxo-1H-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose.Gemcitabine is disclosed in U.S. Pat. Nos. 4,808,614 and 5,464,826,which are incorporated herein by reference for their teaching of how tosynthesize, formulate, and use gemcitabine for treating susceptibleneoplasms. The commercial formulation of gemcitabine hydrochloride isindicated as first-line treatment for patients with locally advanced(nonresectable Stage II or Stage III) or metastatic (Stage IV)adenocarcinoma of the pancreas, and is commonly used in patientspreviously treated with 5-fluorouracil.

[0006] Capecitabine is a fluoropyrimidine carbamate with antineoplasticactivity. It is an orally administered systemic prodrug of5′-deoxy-5-fluorouridine (5′-DFUR) which is converted to 5-fluorouracil.The chemical name for capecitabine is5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine. It is marketed inthe United States as Xeloda™ (Roche Laboratories). It is indicated forthe treatment of patients with metastatic breast cancer and colorectaltumors. It generally is administered for 14 days, followed by a 7-dayrest period during each 21-day cycle. Capecitabine is described in U.S.Pat. No. 5,472,949.

[0007] Cisplatin is the generic name for cis-diaminodichloroplatinum andis described in U.S. Pat. No. 5,562,925, which is incorporated herein byreference. Cisplatin is commercially available and is indicated formetastatic testicular and ovarian tumors, as well as advanced bladdercancer.

[0008] An object of this invention is to provide a method for treatingcancers, especially advanced solid tumors, with a combination comprisingacetyldinaline together with either gemcitabine, capecitabine, orcisplatin. A further object is to provide a composition comprisingsynergistic amounts of acetyldinaline and gemcitabine, synergisticamounts of acetyldinaline and capecitabine, and synergistic amounts ofacetyldinaline and cisplatin.

SUMMARY OF THE INVENTION

[0009] This invention relates to a synergistic combination ofantineoplastic agents, and to a method for treating tumors comprisingadminstering the combination. The invention more particularly provides acomposition comprising, as a first component, acetyldinaline, and as asecond component, either gemcitabine, capecitabine, or cisplatin. Thegemcitabine is used preferably as a pharmaceutically acceptable acidaddition salt.

[0010] The compositions of this invention consist essentially of theabove active ingredients, or suitable salts thereof, together withcommon excipients, diluents, and carriers.

[0011] A preferred composition comprises acetyldinaline, together withgemcitabine hydrochloride. Another preferred combination isacetyldinaline and cisplatin.

[0012] In a further embodiment of the invention, we provide a method fortreating cancer comprising administering to an animal in need oftreatment an effective amount of a combination of acetyldinaline andeither cisplatin, or gemcitabine, preferably as a salt such as thehydrochloride.

[0013] A preferred method embraces treatment of solid tumors.

[0014] A further preferred method employs an antitumor amount ofacetyldinaline and an effective amount of gemcitabine hydrochloride,capecitabine, or cisplatin to treat susceptible cancers, includingnonsmall cell lung cancer (NSCLC), breast cancer, ovarian cancer, headand neck cancer, myelomas, prostate cancer, and pancreatic cancer.

[0015] Another embodiment of the invention is a kit comprising in onecompartment a dosage of acetyldinaline, and in another compartment adosage of gemcitabine, or a pharmaceutically acceptable salt thereof. Afurther embodiment is a kit comprising in one compartment a dosage ofacetyldinaline, and in another compartment a dosage of capecitabine orcisplatin.

DESCRIPTION OF FIGURES

[0016]FIG. 1 shows the reduction in the rate of growth of transplantedtumors in mice following various oral doses of acetyldinaline (CI-994).

[0017]FIG. 2 shows the reduction in the rate of growth of transplantedtumors in mice following various interperitoneal injections ofgemcitabine hydrochloride.

[0018]FIG. 3 shows the reduction in the rate of growth of transplantedtumors in mice following various dose combinations of acetyldinaline andgemcitabine hydrochloride (both agents given at the same time).

[0019]FIG. 4 shows the reduction in the rate of growth of transplantedtumors in mice following various dose combinations of acetyldinaline andgemcitabine hydrochloride (gemcitabine is given first, followed bydosing with CI-994).

[0020]FIG. 5 shows the synergy of CI-994 and gemcitabine in humanovarian carcinoma cells.

[0021]FIG. 6 shows the synergy of CI-994 and cisplatin in human ovariancarcinoma cancer.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The compounds to be utilized in the method of this invention willbe administered in doses commonly employed clinically. Such doses willbe calculated in the normal fashion, for example on body surface area.Acetyldinaline will be administered, for example, at doses from about1.0 mg/m² to about 50 mg/m², preferably from about 2.0 mg/m² to about10.0 mg/m². Ideally, acetyldinaline will be administered at a dose whichwill produce plasma levels of about 5 to about 100 μg/mL. Acetyldinalinetypically is administered orally, for example, as capsules having activeingredient in the amounts of 2.5, 5, and 25 mg per capsule.Acetyldinaline will be administered daily at about the same dose levelsthroughout a treatment period, typically for 15 to 30 days. Multipletreatment periods can be practiced, as dictated by the attending medicalpractitioner and the particular patient and condition being treated.

[0023] Gemcitabine will likewise be administered at doses comparable tothose routinely utilized clinically. For example, the initial dose ofgemcitabine, typically as the hydrochloride salt, will be about 1000mg/m² of body surface area. This product is routinely formulated as asterile solution and is administered by intravenous infusion, generallyover about a 30-minute period, with about 2 to 4 weekly doses, withcourses repeated about every 28 to 30 days. The dose of 1000 mg/m² canbe given for up to about 7 weeks, according to this treatment regimen,or until undesirable side effects are observed. Other salt forms can beutilized if desired, for example, the hydrobromide, monophosphate,sulfate, malonate, citrate, and succinate are readily prepared.

[0024] Capecitabine for monotherapy generally is administered orally ata dose of about 2500 mg/m² daily for 2 weeks, followed by a 1-week restperiod. The product is supplied commercially in 150 mg and 500 mgtablets. The tablets are administered at the rate of about 1 to about 4times a day during the treatment period. The daily doses of capecitabinewill range from about 1000 mg/m² to about 3500 mg/m² per day in thecombinations of this invention.

[0025] Cisplatin generally is formulated as a sterile solution forinjection, and is routinely administered at doses of about 10 to 100mg/m², given intravenously on a daily basis for about 5 to 6 days.Alternatively, cisplatin may be administered as a single dose of about50 to 100 mg/m². This cycle can be repeated for about every 4 to 8weeks.

[0026] The combinations provided by this invention have been evaluatedin several assay systems, and the data can be analyzed utilizing astandard program for quantifying synergism, additivism, and antagonismamong anticancer agents. The program preferably utilized is thatdescribed by Chou and Talalay, in “New Avenues in Developmental CancerChemotherapy,” Academic Press, 1987, Chapter 2.

[0027] The method is based on the median-effect principle of themass-action law using an enzyme kinetic system as a model. The equationis simple and describes the relationships between dose and effectregardless of the shape of the dose-effect curve. Two basic equationsconstitute the pillars of this methodology. To relate dose and effectfor a single drug in the simplest way possible, the median-effectequation derived by Chou is given by:

f _(a) /f _(u)=(D/D _(m))^(m)

D=D _(m) [f _(a)/(1−f _(a))]^(1/m)

[0028] where the right side represents the dose and the left siderepresents the effect, in which f_(a) and f_(u) are the fractionsaffected and unaffected, respectively, D is the dose, D_(m) is themedian-effect dose signifying the potency, and m is a coefficientsignifying the shape of the dose-effect curve. From this equation Chouand Talalay derived the general equation for two or more drugs:$\begin{matrix}{\left\lbrack \frac{\left( f_{a} \right)_{1,2}}{\left( f_{u} \right)_{1,2}} \right\rbrack^{1/m} = \quad {{\left\lbrack \frac{\left( f_{a} \right)_{1}}{\left( f_{u} \right)_{1}} \right\rbrack^{1/m} + \left\lbrack \frac{\left( f_{a} \right)_{2}}{\left( f_{u} \right)_{2}} \right\rbrack^{1/m}} = {+ {\alpha \left\lbrack \frac{\left( f_{a} \right)_{1}\left( f_{a} \right)_{2}}{\left( f_{u} \right)_{1}\left( f_{u} \right)_{2}} \right\rbrack}^{1/m}}}} \\{= \quad {{\frac{(D)_{1}}{\left( D_{m} \right)_{1}} + \frac{(D)_{2}}{\left( D_{m} \right)_{1}}} = \frac{{\alpha (D)}_{1}(D)_{2}}{\left( D_{m} \right)_{1}\left( D_{m} \right)_{2}}}}\end{matrix}$

[0029] where m=1 is for first-order Michaelis-Menten-type kinetics andm>1 (or m<1) is for higher order (or lower order) Hill-type kinetics.When alpha=0, the third term on the right side disappears and whenalpha=1, the third term is conserved. Alpha=0 is used for mutuallyexclusive drugs and alpha=1 is used for mutually nonexclusive drugs. Fordrugs that have the same or similar modes of action, the effects of bothdrugs are mutually exclusive. For drugs that have different modes ofaction or act independently, the effects of both drugs are mutuallynonexclusive.

[0030] A plot of fraction affected (F_(a)) versus combination index (CI)is called the F_(a)-CI plot. This plot indicates synergism, additivity,or antagonism of two drugs at various effect levels in a mixture that isserially diluted. If several mixtures are made, it is possible toestimate the optimal combination ratio for maximal synergy. Differenteffect levels usually give different degress of synergism, additivism,or antagonism. Cl values <1 indicate synergism; Cl values >1 indicateantagonism, and CI values that are one or hover around one indicateadditivity. For anticancer agents, synergism at high effect levels(F_(a)) is clinically more relevant than synergism at low F_(a) levels.

[0031] Gemcitabine, as the monohydrochloride, is routinely usedclinically, and is well-suited as a single agent as a front-linetreatment of patients with advanced pancreatic cancer or NSCLC. It alsois routinely used in combination with other known antineoplastic agents,most notably cisplatin. No synergistic combinations have, however,heretofore been reported.

[0032] While acetyldinaline (CI-994) has not been approved for clinicaluse, it has nevertheless been evaluated in several clinical trials. Inone such study, patients were treated using a dose-escalation schemethat increased both the daily dose and the duration of treatment. Themajority of patients had received extensive prior chemotherapy. Themaximum tolerated dose (MTD) was 15 mg/m²/day when the duration oftreatment was 14 consecutive days. To allow more prolonged treatment,lower doses were studied. Using a schedule of 8 weeks of continuousdaily therapy, followed by a 2-week ‘drug holiday’, the MTD was 8mg/m²/day. The dose-limiting toxicity was thrombocytopenia orneutropenia, usually occurring within 1 month of the start of therapy.Blood counts tended to stabilize even with continued treatment and torecover quickly when treatment was stopped. There was no evidence ofcumulative toxicity following repeated courses and prolonged exposuresto CI-994. Other toxicities included nausea, vomiting, diarrhea,anorexia, fatigue, mucositis, headache, dehydration, and increases inliver and renal function test values. Responses included one partialresponse in a heavily pretreated patient with NSCLC and a minor responsein one patient each with renal cell cancer and NSCLC.

[0033] An additional Phase 1 study was conducted in patients withrelapsed acute leukemia or other hematologic malignancy using a oncedaily high-dose 5-day dosing schedule. The MTD was 135 mg/m²/day. Thedose-limiting toxicity was acute CNS toxicity manifested as sedation andsomnolence. Other adverse events included nausea, vomiting, hypotensionresulting from dehydration, hypocalcemia, headache, and in one patienteach, acute pancreatitis, a pyramidal syndrome characterized byhyperreflexia and bilateral Babinski reflexes, and sepsis. Hematologictoxicities cannot be assessed in this patient population. Two patientswith AML developed tumor lysis syndrome, resulting in one death.Transient decreases in peripheral white blood cell counts were noted.

[0034] A Phase 2 program is currently being conducted with CI-994, usedas a single agent. The dosing regimen is 8 mg/m² given orally daily.Over 100 patients have been treated, including patients with nonsmallcell lung cancer, renal cell cancer, pancreatic cancer, head and neckcancer, ovarian cancer, myeloma, prostate cancer, and breast cancer.Some patients have tolerated dose increases to 10 mg/m², while somepatients have had to have treatment interrupted due to thrombocytopenia,and then be restarted on CI-994 at lowered doses. The adverse eventshave been similar to those observed in the chronic dosing Phase 1protocol. Thrombocytopenia has been the dose-limiting toxicity.Infrequent neurologic adverse events including paresthesias, confusion,and hallucinations have been reported. Objective responses have beenseen in patients with nonsmall cell lung cancer. Clinical benefit hasbeen reported in patients with renal cell cancer.

[0035] In the solid tumor Phase 1 study, CI-994 doses were administeredorally following a fasting period, and blood samples were collected forpharmacokinetic analyses. Preliminary results indicate that the maximumblood level is achieved approximately 1 to 2 hours after ingestion, andthe terminal elimination half-life of CI-994 is approximately 15 hours.The maximum plasma CI-994 concentrations achieved with increasing doselevels were less than dose-proportional. The terminal eliminationhalf-life and the apparent clearance rate were independent of the doseadministered.

[0036] One additional objective of this study was to determine whethertaking CI-994 with food affected its rate or degree of absorption.Twelve fasted patients were given a single dose of CI-994, 8 mg/m². Oneweek later, the same patients were given the same dose of CI-994 with anormal meal. Analysis of pharmacokinetic data revealed that CI-994 canbe taken without regard to meals.

[0037] Mass balance/route of elimination studies have not been conductedin humans. Animal studies indicate that the principal route ofelimination is via renal excretion, with 80% and 62% of radiolabeleddrug appearing in the urine of monkeys and rats, respectively, within 24hours.

[0038] The following detailed examples further establish the synergybetween CI-994 and either gemcitabine, capecitabine, or cisplatin.

EXAMPLE 1

[0039] The synergistic combinations provided by this invention have beenevaluated in standard chemotherapy studies using female BALB/C miceweighing 18 to 20 grams. On Day 0 of the test, each mouse was surgicallyimplanted (subcutaneously) with a fragment of LC-12 squamous cell lungcarcinoma tumor weighing approximately 30 mg. The mice were weighedweekly, and tumor size (width and length in mm) were measured threetimes each week with standard calipers. Tumor mass for each animal wascalculated according to the formula:${{mass} = \frac{\left( {a \times b^{2}} \right)}{2}},$

[0040] where “a” is width of the tumor in mm, and “b” is the length inmm. Evaluation of anticancer activity was established by the formulaT-C, where “T” and “C” are the median time (in days) required for thetreated and control (respectively) tumors to reach a pre-determined sizeof 750 mg (the “evaluation size”). Acetyldinaline was suspended in 0.5%aqueous methyl cellulose and administered orally at various dosages in0.5 mL volumes. Gemcitabine hydrochloride was dissolved in 5% aqueoussaline and administered intraperitoneally at various dosage levels in0.5 mL injections.

[0041] The animals were divided into four groups. One group served ascontrols and received no drug treatments. One group (FIG. 1) was furtherdivided into four sub-groups, each of which received oral doses ofacetyldinaline at a specified level of active drug (15 mg/kg, 30 mg/kg,45 mg/kg, and 60 mg/kg). The acetyldinaline was administered daily onDays 10-14 (Day 0 being when the tumor was implanted), Days 17-21, andDays 24-28. FIG. 1 shows that acetyldinaline delays the growth of theimplanted tumor at all dose levels, relative to untreated controls.Tumors in untreated controls exhibited significant growth, starting onDay 10. At the 30 and 45 mg/kg dosages of acetyldinaline, significanttumor growth did not commence until about Day 24. At 60 mg/kg, growthwas delayed for 33 days.

[0042]FIG. 2 shows the effects of gemcitabine hydrochloride at 40 and 80mg/kg, administered IP on Days 10, 13, 16, and 19. Tumor growth wassubstantially inhibited for 25 days at 40 mg/kg, and for about 32 daysat 80 mg/kg. (Controls showed significant growth starting at Day 10.)

[0043]FIG. 3 shows the effects of various dose combinations ofacetyldinaline and gemcitabine hydrochloride (e.g., 15 mg/kg ofacetyldinaline with 40 mg/kg gemcitabine hydrochloride; 15/80, 30/40,30/80, and 45/20). The test animals were implanted with an LC 12squamous cell lung carcinoma, and were divided into four sub-groups,each sub-group receiving a different dosage of combination therapy.Acetyldinaline was administered orally at the indicated dosages on Days10-14 (e.g., 15 mg/kg daily), Days 17-21, and Days 24-28. Thegemcitabine hydrochloride was administered IP on Days 10, 13, 16, and19. The results in FIG. 3 establish a dramatic and synergistic reductionin tumor growth for all combination doses of drugs. At the combinationof 15 mg/kg acetyldinaline and 40 mg/kg gemcitabine, no substantialgrowth of the implanted tumor occurred until Day 28 (tumors in controlsstarted substantial growth on Day 10). At combination doses of 15/80,30/40, 30/80, and 45/20 (acetyldinaline/gemcitabine), tumor growth wassubstantially inhibited for the full 60-day period of the test.

[0044]FIG. 4 shows the results of a combination study carried out for 75days. All test animals were implanted with an LC12 squamous cell lungcarcinoma. Animals selected to receive test drugs were divided into foursub-groups. One sub-group received 15 mg/kg of acetyldinaline incombination with 80 mg/kg of gemcitabine. A second sub-group receivedthe combination at a dose of 15 mg/kg/160 mg/kg. The third subgroupreceived the combination at a dose of 30 mg/kg acetyldinaline with 80mg/kg of gemcitabine. The fourth sub-group received the combination of45 mg/kg/80 mg/kg.

[0045] The acetyldinaline was administered orally to each test group onDays 26-30, 33-37, and 40-44. Gemcitabine was administered IP on Days10, 13, 16, and 19.

[0046]FIG. 4 shows that the tumors in the untreated controls startedgrowing by the 11^(th) day following implantation, and reached 750 mg(the “evaluation size”) on Day 18. In contrast, tumors in animalstreated with 15 mg/kg acetyldinaline and 80 mg/kg of gemcitabine showedvery little growth until Day 35, and did not reach evaluation size untilDay 42. Tumors in animals dosed with the 30 mg/kg/80 mg/kg combinationstarted growing at Day 35, but did not reach the 750 mg evaluation sizeuntil Day 52. The tumors in animals dosed with the 15 mg/kg/160 mg/kgcombination, as well as the 45 mg/kg/80 mg/kg combination, showed nogrowth, even at Day 75 when the test was ended.

[0047] The data presented in FIGS. 2 and 4 establish that thecombination of acetyldinaline and gemcitabine is surprisingly active inreducing the rate of growth of tumors in animals. The ability of theseagents when used together establish the combination to be synergistic asan antitumor agent.

EXAMPLE 2

[0048] Because the synergistic effects observed with the combination ofacetyldinaline and gemcitabine (shown in FIGS. 2 and 4) were sosurprisingly dramatic, the experiment described above in Example 1 wasrepeated with additional doses of acetyldinaline. Table 1 below showsthe activity of CI-994 and gemcitabine, alone and in combination, in theLC-12 mouse lung tumor model when tested according to the procedure ofExample 1. TABLE 1 Antitumor Effect of the Combination of CI-994 andGemcitabine in the LC-12 Mouse Lung Tumor Model CI-994 Gemcitabine Toxic% Weight Antitumor Effect Dose^(a) Schedule Dose^(a) Schedule DeathsChange^(b) CR^(c) PR^(d) T-C^(e) Net Log₁₀ Kill^(f) 7.5 11-15, 18-22,25-29 0 — 0/6 +4 1/6 0/6 2.4 −1.2 15 0 — 0/6 +10 0/6 0/6 0.4 −1.4 30 0 —0/6 +1 0/6 0/6 1.6 −1.3 60 0 — 0/6 −9 3/6 0/6 7.8 −0.8 0 — 80 11, 14,17, 20 0/6 +1 1/6 0/6 15.4 0.5 0 — 160 0/6 −3 0/6 1/6 24.0 1.2 7.511-15, 18-22, 25-29 80 11, 14, 17, 20 0/6 +8 2/6 0/6 23.5 0.4 15 80 0/6+7 2/6 1/6 23.8 0.5 30 80 0/6 +8 5/6 0/6 37.0 1.5 60 80 3/6 −8 ToxicToxic Toxic Toxic 7.5 11-15, 18-22, 25-29 160 11, 14, 17, 20 0/6 −8 3/60/6 25.6 0.6 15 160 0/6 −3 4/6 1/6 29.8 0.9 30 160 0/6 −3 5/61/6 >82.6 >5.0 60 160 0/6 — Toxic Toxic Toxic Toxic

EXAMPLE 3

[0049] The procedure of Example 1 was followed to evaluate theanti-tumor activity of combinations of CI-994 and cisplatin compared tothe individual agents alone. The results are presented in Table 2. Thetable shows that treatment with 60 mg/kg/day of CI-994 caused twocomplete responses out of 10 test animals, while cisplatin alone at 2mg/kg/day failed to cause any complete responses. The combination,however, caused a 70% complete response, namely complete responses in 7out of 10 treated animals. The T-C effect was even more dramatic, goingfrom 8.7 and 2.4 for the monotherapy treatments, to 84.2 for thecombination treatment. TABLE 2 Antitumor Effect of the Combination ofCI-994 and Cisplatin in the LC-12 Mouse Lung Tumor Model CI-994Gemcitabine Toxic % Weight Antitumor Effect Dose^(a) Schedule Dose^(a)Schedule Deaths Change^(b) CR^(c) PR^(d) T-C^(e) Net Log₁₀ Kill^(f) 0 —0 — 0/10 +15 0/10 0/10 — — 7.5 11-15, 18-22, 25-29 0 — 0/10 +9 0/10 0/10−1.4 0 15 0 — 0/10 +11 0/10 0/10 −1.9 0 30 0 — 0/10 +11 0/10 0/10 4.3−0.8 60 0 — 0/10 +11 2/10 0/10 8.7 −0.5 0 — 1 11-15 0/10 +13 0/10 0/102.6 −0.1 0 — 2 0/10 +12 0/10 0/10 2.4 −0.1 7.5 11-15, 18-22, 25-29 111-15 0/10 +4 0/10 0/10 2.0 −0.9 15 1 0/10 +4 1/10 0/10 8.2 −0.6 30 10/10 +3 4/10 2/10 6.7 −0.7 60 1 0/10 +2 7/10 1/10 12.7 −0.3 7.5 11-15,18-22, 25-29 2 11-15 0/10 +1 1/10 0/10 10.0 −0.5 15 2 0/10 −2 3/10 0/1017.0 −0.1 30 2 0/10 −1 5/10 1/10 20.6 0.2 60 2 0/10 −14 7/10 3/10 84.23.8

EXAMPLE 4

[0050] The general procedure of Example 1 was followed to evaluateCI-994 and capecitabine, alone and in combination, against a colontumor. The assay was carried out the same as described in Example 1,except that a fragment of C26 colon tumor was surgically implanted intothe mice, instead of the LC-12 lung tumor which was used in theforegoing examples. Table 3 reports the results of monotherapy withspecific doses of CI-994, and with capecitabine (Xeloda™), and withcombination therapy wherein the animals were first dosed withcapecitabine (on Days 3-7 and 10-14), followed by dosing with CI-994 (onDays 17-21, 24-28, and 31-35). The data in Table 3 establish that noneof the animals receiving CI-994 alone were tumor free at the end of theassay, that 30% (3 out of 10) of the animals receiving capecitabinealone were tumor free, and that 70% (7 out of 10) animals receiving thecombination of CI-994 plus capecitabine were tumor free.

[0051] Table 4 presents the results of the same assay, except that thecombination agents, CI-994 and capecitabine, both were administeredsimultaneously on Days 3-7 and 10-14. Dosing with CI-994 was continuedon Days 17-21, whereas capecitabine therapy was stopped at Day 14. TABLE3 Antitumor Effect of the Combination of CI-994 and Xeloda in the C26Mouse Colon Tumor Model: Effect of Treatment with Xeloda Followed byTreatment with CI-994 against Early Stage Tumor CI-994 Xeloda Toxic %Weight Antitumor Effect Dose^(a) Schedule Dose^(a) Schedule DeathsChange^(b) CR^(c) PR^(d) T-C^(e) (add.) Net Log₁₀ Kill^(f) % Tumor free7.5 17-21, 24-28, 31-35 0 — 0/10 −5 Early Stage Early Stage  3.1 −1.30/10 15 0 — 0/10 −9 Early Stage Early Stage  6.7 −1.0 0/10 30 0 — 1/10−16 Early Stage Early Stage  8.4 −0.8 0/10 60 0 — 0/10 −10 Early StageEarly Stage 10.1 −0.7 0/10 0 — 500 3-7, 10-14 0/10 −0.2 Early StageEarly Stage 13.8 0.2 2/10 0 — 750 1/10 −18 Early Stage Early Stage 20.70.8 3/10 7.5 17-21, 24-28, 31-35 500 3-7, 10-14 0/10 −1 Early StageEarly Stage 13.7 (16.9) −0.4 3/10 15 500 0/10 −1 Early Stage Early Stage17.3 (20.5) −0.1 4/10 30 500 0/10 −4 Early Stage Early Stage 18.3 (22.2)0.0 3/10 60 500 0/10 −4 Early Stage Early Stage 20.4 (23.9) 0.2 6/10 7.517-21, 24-28, 31-35 750 3-7, 10-14 1/10 −13 Early Stage Early Stage 19.1(23.8) 0.1 4/10 15 750  2/10^(g) −15 Early Stage Early Stage 29.8 (27.1)0.4 2/10 30 750 1/10 −10 Early Stage Early Stage 45.0 (29.1) 2.3 7/10 60750 6/10 Toxic Early Stage Early Stage Toxic Toxic Toxic

[0052] TABLE 4 Antitumor Effect of the Combination of CI-994 and Xelodain the C26 Mouse Colon Tumor Model: Effect of Simultaneous TreatmentWith CI-994 and Xeloda Against Early Stage Tumor CI-994 Xeloda Toxic %Weight Antitumor Effect Dose^(a) Schedule Dose^(a) Schedule DeathsChange^(b) CR^(c) PR^(d) T-C^(e) (add.) Net Log₁₀ Kill^(f) % Tumor Free7.5 3-7, 10-14, 17-21 0 — 0/10 +5 Early Stage Early Stage  1.6 −2.2 0/1015 0 — 0/9  −8 Early Stage Early Stage  1.0 −2.3 0/9  30 0 — 0/9  −11Early Stage Early Stage  4.6 −1.8 0/9  60 0 — 0/9  −12 Early Stage EarlyStage  4.8 −1.8 0/9  0 — 500 3-7, 10-14 0/10 +2 Early Stage Early Stage15.2 0.6 0/10 0 — 750 0/10 −12 Early Stage Early Stage 20.4 1.3 1/10 7.53-7, 10-14, 17-21 500 3-7, 10-14 0/10 +6 Early Stage Early Stage 15.4(16.8) −0.4 0/10 15 500 0/10 +6 Early Stage Early Stage 14.5 (16.2) −0.51/10 30 500 0/10 −11 Early Stage Early Stage 14.2 (19.8) −0.5 0/10 60500 0/10 −8 Early Stage Early Stage 18.1 (20.0) 0.0 1/10 7.5 3-7, 10-14,17-21 750 3-7, 10-14 0/10 −11 Early Stage Early Stage 19.1 (22.0) 0.20/10 15 750 0/10 −7 Early Stage Early Stage 16.9 (21.4) −0.2 2/10 30 7500/10 −14 Early Stage Early Stage 17.4 (25.0) −0.1 0/10 60 750 1/10 −18Early Stage Early Stage 16.4 (25.2) −0.2 0/10 Early Stage Early Stage

EXAMPLE 5

[0053] The combinations of CI-994 plus gemcitabine, and of CI-994 pluscisplatin, were evaluated in human ovarian carcinoma cells, and the datawas analyzed according to the Chou and Talalay program which establishedboth combinations to be synergistic.

[0054] Ovcar-4 Human Ovarian

[0055] Ovcar-4 human ovarian carcinoma cells were seeded into 96-wellculture plates in RPMI 1640 culture media supplemented with 20% fetalcalf serum and 10 μg/mL of insulin. Various concentrations of CI-994 andeither cisplatin or gemcitabine were added together 24 hours after cellswere initially seeded into the culture plates and allowed to attach. Theeffect of CI-994, gemcitabine or cisplatin alone and in combination onOvcar-4 proliferation was determined after 96 hours of incubation at 37°C. using the SRB assay as previously described (Skehan P., Stoneng R,Scudiero D, et al. New colorimetric cytotoxicity assay foranticancer-drug screening. J. Natl. Cancer Inst. 1990;82:1107-1112). Thecombination chemotherapy data was analyzed using the Biosoft program,“Dose Effect Analysis with Microcomputers for IBM PC”, which is astandard program for quantifying synergism, additivism, and antagonismamong anticancer agents, and is based on the median-effect principle ofmass-action law using an enzyme kinetic system model described by Chouand Talalay. Plots of fraction affected (F_(a)) versus combination index(CI) are called Fa-CI plots. These plots indicate synergism, additivity,or antagonism of 2 drugs at various effect levels in a mixture that isserially diluted. If several mixture are made, it is possible toestimate the optimal combination ratio for maximal synergy. Differenteffect levels usually give different degrees of synergism, additivism,or antagonism. CI values <1 indicate synergism; CI values >1 indicateantagonism and values that hover around 1 as a straight line indicateadditivity. FIGS. 5 and 6 show representative Fa-CI plots for CI-994plus gemcitabine (FIG. 5), and CI-994 plus cisplatin (FIG. 6). In bothplots, CI values over the entire Fa range are less than one, indicatingsynergy for the drug combinations.

Clinical Evaluation of Combination Therapy

[0056] This is a multicenter, open-label Phase 1 study of CI-994 givenin combination with gemcitabine to patients with advanced solid tumors.

[0057] The objectives of this study are to determine the, (1) maximumtolerated dose (MTD), (2) recommended Phase 2 dose, (3)pharmacokinetics, (4) safety profile, and (5) to observe for antitumoractivity of CI-994 when given in combination with gemcitabine topatients with advanced solid tumors. The primary efficacy endpoint isthe attainment of either a partial response (PR) or a complete response(CR). Secondary endpoints include time to PR or CR, duration of PR orCR, and survival.

[0058] Gemcitabine is administered as a 30-minute intravenous infusionon Days 1, 8, and 15 of a 28-day treatment course, using an initial doseof 1000 mg/m². CI-994 is administered orally as a daily dose for 21 daysof a 28-day course, beginning on Day 1. Patients may receive subsequentcourses of treatment based on individual tolerance and response totherapy. Patients whose disease does not respond or who developintolerable adverse events are discontinued from study treatment.

[0059] The initial dose level of CI-994 is' 4 mg/m². A minimum of 3patients will be treated at each dose level. Dose levels are increasedby 2 mg/m² until the MTD is reached. Ten additional patients are to betreated at the dose level recommended for Phase 2 studies, which isexpected to be the MTD or one dose level below the MTD.

[0060] Once a patient begins study treatment, the addition of othercancer treatment will confound the assessment of safety and efficacy andtherefore is not allowed. This restriction precludes the addition ofsystemic cytotoxic, hormonal, immunologic, or other biologic agentswhile the patient is in the treatment phase of this protocol. Patientswho require palliative radiotherapy while on study are generallyconsidered to have progressive disease and, unless compellinginformation exists to the contrary, are to be discontinued from studymedication. Patients who develop new brain metastases while on study mayhave treatment interrupted to receive a course of cranial irradiation,then be restarted on study medication after a recovery period of atleast 1 week.

[0061] Antiemetics may be used at the investigator's discretion forprevention and/or treatment of nausea or vomiting. Every effort shouldbe made to ensure nausea and vomiting is controlled, as these conditionsmay preclude a patient from taking or absorbing the oral doses ofCI-994. This issue is particularly relevant on Days 1, 8, and 15, whengemcitabine is also administered. Because CI-994 caused sedation andsomnolence at higher doses in the Phase 1 program, antiemetics that areleast likely to cause these side effects should be used.

[0062] Colony-stimulating factors may be used at the investigator'sdiscretion to treat episodes of severe myelosuppression that arecomplicated by infection, but should otherwise not be used to supportlow blood counts or to maintain dose intensity.

[0063] If criteria are met for a Complete Response (CR), administer 2additional courses of treatment beyond confirmation of the CR and thencompletely reassess the patient's disease state. If the patient isconsidered to be clinically free of disease at that time, discontinuethe gemcitabine and continue to administer CI-994 for 3 additionalmonths, using the same dose and schedule (3 weeks on/1 week off). Atthat time, again completely reassess the patient's disease state. If thepatient is still in CR, the investigator must evaluate the risks andpotential benefits of continuing CI-994 treatment.

[0064] Treatment Courses

[0065] A treatment course consists of gemcitabine given intravenously onDays 1, 8, and 15 of a 28-day course plus CI-994 administered dailyorally, beginning on Day 1, for 21 days of a 28-day course. Courses areto be repeated on Day 29 if there has been adequate recovery fromadverse events and myelosuppression, defined as nonhematologicparameters of Grade ≧1, platelet count ≧100,000 μL, and absoluteneutrophil count ≧1500μL. Subsequent courses may be delayed by weeklyintervals up to 3 weeks. If recovery has not occurred by Day 50, thepatient is to be discontinued from study medication.

[0066] Gemcitabine Dosing

[0067] The initial dose of gemcitabine in each course is 1000 mg/m²,given as a 30-minute intravenous infusion. Dose adjustments may berequired during a treatment course. Follow the manufacturer'srecommendations for information regarding preparation andadministration. CI-994 Dose Levels CI-994 doses are calculated based onbody surface area (BSA) and must then be rounded to the closestavailable capsule strength. CI-994 is available in capsule strengths of2.5, 5, and 25 mg. Doses may be taken without regard to meals.

[0068] The initial CI-994 dose level is 4 mg/m². Subsequent dose levelswill be increased (or decreased if necessary) by a fixed increment of 2mg/m² until the MTD is determined. Individual patients may not receivedose escalations of gemcitabine or CI-994 in subsequent courses.Patients may receive a lower dose of CI-994 in a subsequent course ifdose-limiting toxicities were experienced.

[0069] Three new patients will be assessed at each new dose level. Theminimum time that these patients must be followed is 4 weeks before anew dose level may be opened (unless treatment was interrupted earlierand the patient is recovering from adverse events). If none of these 3patients experience a dose-limiting toxicity, the next higher dose levelwill be opened. If one patient develops a dose-limiting toxicity, 3 morepatients will be enrolled at that dose level. If>2 of 6 patientsexperience a dose-limiting toxicity at the same level, that dose levelwill be considered the MTD.

[0070] An assessable patient is defined as one who received 3 weeklydoses of gemcitabine plus at least 80% of the CI-994 doses (>17 doses),or a patient whose treatment course was discontinued early or wasnoncompliant (<17 doses) due to treatment-related adverse events. Apatient who took fewer than 17 doses of CI-994 or did not complete thetreatment course because of nontreatment-related reasons (eg, missedappointments, ran out of CI-994 supplies, developed a coexisting medicalcondition that rendered the patient unable to swallow capsules,developed rapidly progressing disease) is not considered to be anassessable patient for the tolerability of that dose level.

[0071] Patients should be encouraged to take their CI-994 dose atapproximately the same time each day. However, a variance of up to 12hours either way is allowed for any given dose, rather than miss a day'sdose. If a patient misses a day's dose entirely, they must be instructednot to ‘make it up’ the next day. If a patient vomits anytime aftertaking a dose of CI-994, they must be instructed not to ‘make it up’,but to resume subsequent doses the next day as prescribed.

[0072] On Days 1, 8, and 15, the CI-994 dose should be given 2 hoursbefore the gemcitabine dose, to ensure maximal absorption in the eventthe patient develops vomiting following the gemcitabine dose.

[0073] Once the MTD is determined, 10 additional patients will betreated at the Phase 2 dose level, which is expected to be the MTD orone dose level below the MTD.

[0074] Dose Adjustments During a Course

[0075] Continuation of gemcitabine and CI-994 during a course isdependent on patient tolerance and hematologic parameters. Reduced dosesof gemcitabine may be required on Days 8 and 15, as recommended by themanufacturer and shown in the table below. The dose of CI-994 is not tobe increased or decreased during a treatment course, although earlytermination may be required as described below. If both studymedications must be stopped before a course is completed, do notcomplete that course, but instead follow the patient for recovery, thenstart another course using a reduced dose of CI-994. Dose AdjustmentsDuring a Treatment Course % Full Dose of % Full Dose of ConditionGemcitabine CI-994 Absolute Neutrophil Count/μL ≧1000 100 100 ≧500 to999  75 100 <500  0  0 -OR- Platelet Count/μL ≧100,000 100 100 ≧50,000to 99,000  75 100 <50,000  0  0 -OR- Treatment-Related NonhematologicToxicity Grade 0-2 100 100 Grade 3 or 4  0  0

[0076] Gemcitabine dose adjustments shown in the table above are basedon the results on that day. For example, on Day 8 draw a CBC and checkthe results. Determine if dosing is to continue. If so, have the patienttake the CI-994 dose. Two hours later, give the gemcitabine dose which,if necessary, has been reduced. The decision to discontinue CI-994dosing during a course is based on adverse events or hematology resultsat any time. Example: A patient has a platelet count on Day 11 of 45,000μL. Instruct the patient to stop taking CI-994 capsules (and to returnall study medication containers to the site). Obtain another CBC on Day15. If the platelet count on Day 15 is 50,000 to 99,000μL, administer75% of the calculated gemcitabine dose but do not reinstitute CI-994dosing. If the platelet count on Day 15 remains below 50,000 μL, do notretreat with gemcitabine. Consider this course to be terminated andfollow the patient for recovery. In either case, the patient may receivea subsequent treatment course using the same initial dose of gemcitabine(1000 mg/m²) and a CI-994 dose that has been reduced by 2 mg/m².

[0077] Drug Formulation and Stability

[0078] Gemcitabine is to be obtained by the site from commercialsources. Follow the manufacturer's recommendation for preparation,administration, stability, and storage conditions.

[0079] CI-994 is formulated in identically appearing gelatin capsulescontaining 2.5, 5, or 25 mg of study medication, plus inactiveingredients of lactose, cornstarch, and talc or polyethylene glycol6000. Store at controlled room temperature.

[0080] The foregoing data establish an unexpectedly favorableinteraction between acetyldinaline and gemcitabine, and betweenacetyldinaline and cisplatin. Accordingly, this invention provides amethod of treating susceptible neoplasms comprising administeringacetyldinaline in a regimen together with either gemcitabine, apharmaceutically acceptable salt thereof, or cisplatin. The combinationgenerally will include each active ingredient packaged separately,thereby avoiding any interaction between the agents prior toadministration. If desired, the individually packaged drugs can beplaced in a single carton as a kit, thereby providing convenience to theattending physician or medical attendant. The susceptible neoplasms tobe treated according to this invention include solid tumors, especiallyadvanced solid tumors and nonsmall cell lung cancer, as well as renalcell cancer, pancreatic cancer, head and neck cancer, ovarian cancer,myeloma, prostate cancer, and breast cancer.

What is claimed is:
 1. A combination of antineoplastic agents comprisingan antitumor amount of acetyldinaline and an antitumor amount ofgemcitabine, a pharmaceutically acceptable salt thereof, capecitabine,or cisplatin.
 2. A combination of claim 1 comprising acetyldinalineformulated as a capsule.
 3. A combination of claim 2 comprisinggemcitabine or a pharmaceutically acceptable salt thereof formulated asa sterile solution for intravenous infusion.
 4. A combination of claim 3comprising gemcitabine monohydrochloride.
 5. A combination of claim 3comprising gemcitabine monohydrobromide.
 6. A combination of claim 2comprising cisplatin.
 7. A combination of claim 2 comprisingcapecitabine.
 8. A method of treating cancer comprising administering toan animal in need of treatment an antitumor amount of a combination ofclaim
 1. 9. A method of claim 8 wherein the cancer treated is nonsmallcell lung cancer.
 10. A method of claim 9 comprising administeringacetyldinaline in combination with gemcitabine monohydrochloride.
 11. Amethod of claim 9 comprising administering acetyldinaline in combinationwith cisplatin.
 12. A method of claim 9 comprising administeringacetyldinaline in combination with capecitabine.
 13. A method of claim 8wherein the cancer treated is prostate cancer.
 14. A method of claim 13comprising administering acetyldinaline in combination with gemcitabinemonohydrochloride.
 15. A method of claim 13 comprising administeringacetyldinaline in combination with cisplatin.
 16. A method of claim 13comprising administering acetyldinaline in combination withcapecitabine.
 17. A method of claim 8 wherein the cancer treated is alocally advanced (nonresectable Stage II or Stage III) or metastatic(Stage IV) adenocarcinoma of the pancreas.
 18. A kit comprisingacetyldinaline in one compartment and gemcitabine or a pharmaceuticallyacceptable salt thereof in a second compartment.
 19. A kit comprisingacetyldinaline in one compartment and cisplatin in a second compartment.20. A kit comprising acetyldinaline in one compartment and capecitabinein a second compartment.